1. Field of the Invention
The present invention concerns the field of absorbance detection. Specifically, the present invention concerns an apparatus and method for absorbance detection in instrumental situations which have short absorption path lengths. More specifically, the present invention relates to an analytical method and apparatus that employs a lab-on-a-chip device such as electrophoretic or flow injection type microchips.
2. Discussion of the Background Art
Ultraviolet and visible (UV-VIS) absorbance detection provides one of the most general detection methods in spectrochemical analysis. UV-VIS absorbance detection is often used with microchip basal devices. However, these devices inherently offer shallow channel of about 10-20 microns in depth, thus limiting the path length through which the UV-VIS light can travel. This results in a detection sensitivity which is insufficient for such devices to be of practical use.
Several approaches have been tried to overcome the above mentioned limitations. Harrison et al. (Anal. Chem. 1996, 68, 1040-1046) employed a U-cell which increases path length by allowing the beam to pass in a longitudinal direction along the flow channel. However, this cell requires insertion of optical fibers into etched channels making it difficult to manufacture. Harrison (Electrophoresis 2000, 21 1291-1299) also tried a multipath cell in which lithographically fabricated aluminum mirrors above and below the flow channel formed a multireflection cell. A 633 nm Helium Neon laser beam was launched through an aperture into the cell at a slightly angle. This arrangement yielded a 5 to 10 fold increase of effective optical path length. However, this type of device only works with a collimated laser beams. Thus, this approach is unsuited for incoherent lamp light illumination such as is used in UV-VIS absorbance detection methods.
A further method for improving detection sensitivity in microchip devices is disclosed in European patent application EP 0 840 113 A2, which teaches a method and means for simultaneous detection of migration patterns along a channel using cylinder optics to transmit and collect light transmitted through the channel along separation passages, eventually detecting it by means of a photodiode array (PDA) oriented parallel to the channel. The increased integration time improves signal-to-noise ratio, but with the drawback of increased analysis time.
It is therefore an object of the present invention to provide an apparatus and method for absorbance detection which overcomes the shortcomings as stated above. It is a further object of the present invention to improve the detection limit of UV-VIS absorbance detection in microchip instrumentation.
According to the present invention there is provided an apparatus for measuring absorbance comprising a light source emitting a sample beam which is incident upon a cell having a sample area, the cell being arranged to reflect the sample beam to a detector, characterized in that the apparatus further comprises a modulation means arranged to modulate the sample beam so as to improve the sensitivity of absorbance measurement.
According to an aspect of the present invention, the modulation means includes a scanning device arranged to move the sample beam from a first position in which the sample beam is incident upon said sample area to a second position in which the sample beam is incident upon the cell.
According to a further aspect of the present invention the modulation means may be a linear scanning device. The linear scanning device may be arranged to move the cell. Alternatively, the linear scanning device may be arranged to move an optical element.
In an alternative embodiment of the present invention, the modulation means may be an angular scanning device. The angular scanning device may be a galvanometer.
Advantageously, the present invention increases the analytical sensitivity by doubling optical path length, thus doubling analytical sensitivity, and by reduction of baseline noise by means of modulated or homodyne detection in combination with dual-beam detection for common mode rejection purposes. Doubling of effective optical path length is accomplished by means of reflection type sample light path, while baseline noise reduction is achieved by means of dual-beam detection together with sample modulation and synchronous demodulation. Modulated detection at one certain modulation frequency, also termed homodyne detection, is a proven strategy for recovering small signals buried in asynchronous background and noise. Both homodyne detection, as well as sample modulation, can be implemented with simple circuitry, from example, dual-phase lock-in amplifiers and sample modulation means.
In order to gain the advantages of modulated detection methods, a suitable parameter of measurement needs to be modulated in time periodically with a certain frequency. According to the present invention this is accomplished by a movement of the sample in and out of the sample light path or vice versa using suitable modulation means.
Advantageously, by utilising the back reflection of light launched through the micro channel, the present invention doubles the sensitivity of the apparatus by doubling the absorption path length.
Furthermore, by employing a modulation technique, the baseline noise is reduced, thus further improving the detection limit of the apparatus.